Virginia Tech/plasmid construct

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The Original Plan:
 
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To test our model experimentally, we need to develop a biological system that will determine if a cell goes lytic or lysogenic after infection.
 
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Our construct must contain three important elements:
 
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1) a bistable promoter
 
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2) Green fluorescent protein (GFP)
 
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3) Yellow fluorescent protein (YFP)
 
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The bistable promoter will determine if the cell goes lytic or lysogenic after infection. If the cell goes lytic, enough Cro is produced to fluoresce the cell green. If the cell goes lysogenic, enough CI is produced to fluoresce the cell yellow.
 
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Because a bistable promoter was used, we need to first “flip” GFP.
 
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While trying to “flip” GFP we came across a few technical difficulties. It was believed that YFP had possibly been mislabeled or contaminated. After having similar problems with other registry parts, our lab decided to undertake a sequencing project of the entire registry. We believe that this will help with quality control and help eliminate future problems.
 
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Alternative Plan:
 
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After having difficulties creating the construct, we decided it would be in our best interest to synthesis the construct. The synthesized construct contained commercial parts, dsRED and acGFP instead of YFP and GFP.
 
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[[Image:Gene_circuit_fl.PNG|thumb|right|400px|'''The reporter plasmid''' the two different fluorescent genes are each on opposite sides of the right promoter of the λ switch.]]
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<h3>Our original plan was to build our plasmid in two ways.</h3> We wanted to contrast the iGEM method to more traditional methods of molecular biology. Both approaches required three DNA parts:
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1) A bistable promoter derived from the λ switch (see [[Virginia_Tech/plasmid_design|Designing the Plasmid]])
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2) Two fluorescent protein genes from the registry
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3)      A vector backbone from the registry
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While constructing our plasmid, we decided it would be interesting to sequence the registry in order to add more quality control. Other members of our lab undertook this project. More details are [[Virginia_Tech/sequencing|here]].
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Our more traditional plan involved synthesizing the plasmid. We knew this would be a quicker way to get the plasmid so that we could begin testing it and obtain experimental data for our model. The results from this approach did not turn out as we expected. We were delayed in finishing the design and having it synthesized.
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Latest revision as of 03:24, 27 October 2007

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Constructing the Reporter Plasmid


The reporter plasmid the two different fluorescent genes are each on opposite sides of the right promoter of the λ switch.

Our original plan was to build our plasmid in two ways.

We wanted to contrast the iGEM method to more traditional methods of molecular biology. Both approaches required three DNA parts:

1) A bistable promoter derived from the λ switch (see Designing the Plasmid)

2) Two fluorescent protein genes from the registry

3) A vector backbone from the registry


While constructing our plasmid, we decided it would be interesting to sequence the registry in order to add more quality control. Other members of our lab undertook this project. More details are here.

Our more traditional plan involved synthesizing the plasmid. We knew this would be a quicker way to get the plasmid so that we could begin testing it and obtain experimental data for our model. The results from this approach did not turn out as we expected. We were delayed in finishing the design and having it synthesized.


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